The present invention relates to a method and reagents for a fluorescence polarization immunoassay (FPIA) procedure for determining the amount of flecainide in fluids, especially biological fluids such as serum, plasma or urine, and to a method for making certain of the reagents. More specifically, the invention relates to (1) reagents for determining the amount of flecainide in a sample, viz., tracer and buffer solutions; (2) synthetic methods for preparing the tracer compounds; and (3) analytical methods for conducting the assay.
Flecainide acetate, or N-(2-piperidylmethyl)-2,5-bis (2,2,2-trifluoroethoxy)benzamide acetate, is a drug used to treat ventricular arrhythmias and tachycardia. Although flecainide acetate has been proven to be effective clinically, it can cause undesirable side effects if optimal dosage levels are exceeded. Therapeutic blood flecainide levels have been found to range from 0.2 to 1.0 microgram per milliliter, with 0.5 microgram per milliliter considered the mean effective concentration. Monitoring of serum flecainide levels combined with clinical data can provide the physician with useful information to aid in adjusting patient dosage, achieving optimal therapeutic effects while avoiding useless subtherapeutic or harmful toxic dosage levels.
In the past, patient serum or plasma flecainide levels have typically been measured by high performance liquid chromatography (HPLC) or gas chromatography (GC). Although these methods are reasonably specific for detecting drug levels, they are not without drawbacks. They involve extensive sample preparation, time-consuming instrument set-up and a need for highly trained personnel.
In assays for other substances, competitive binding immunoassays have provided a more satisfactory alternative. Typically, competitive binding immunoassays are used for measuring ligands in a test sample. (For purposes of this disclosure, a "ligand" is a substance of biological interest to be determined quantitatively by a competitive binding immunoassay technique.) The ligands compete with a labeled reagent, or "ligand analog," or "tracer," for a limited number of receptor binding sites on antibodies specific to the ligand and ligand analog. The concentration of ligand in the sample determines the amount of ligand analog which binds to the antibody: the amount of ligand analog that will bind is inversely proportional to the concentration of ligand in the sample, because the ligand and the ligand analog each bind to the antibody in proportion to their respective concentrations.
Fluorescence polarization provides a quantitative means for measuring the amount of tracer-antibody conjugate produced in a competitive binding immunoassay. Fluorescence polarization techniques are based on the principle that a fluorescent labeled compound, when excited by plane polarized light, will emit fluorescence having a degree of polarization inversely related to its rate of rotation. Accordingly, when a tracer-antibody conjugate having a fluorescent label is excited with plane polarized light, the emitted light remains highly polarized because the fluorophore is constrained from rotating between the time that light is absorbed and emitted. In contrast, when an unbound tracer is excited by plane polarized light, its rotation is much faster than that of the corresponding tracer-antibody conjugate. As a result, the light emitted from the unbound tracer molecules is depolarized.
Fluorescence polarization immunoassay techniques are well known in the art, to date, however, the use of such techniques for the determination of flecainide levels have not been successfully attempted. Thus, the present invention offers an advance in the art in that highly sensitive tracers, a method for making the tracers, and an assay using the tracers are provided specifically for the determination of flecainide. The assay conducted in accordance with the present invention is particularly accurate, as will be explained infra.